Structure determination of eukaryotic integral membrane proteins is challenging and techniques and strategies developed recently has underpinned GPCR crystallization, including the development of receptor-T4 lysozyme (T4L) fusions (1,2), conformational thermostabilisation of GPCRs (3-7), and the use of antibody fragments (8-10).
However, the key component for successful crystallization is the stability of the GPCR during purification and crystallization (11). Techniques such as the addition of high affinity ligands to receptor-T4L fusions, and systemic mutagenesis coupled to a thermostability assay (3, 4, 6, 7) have improved the stability of GPCRs in detergent solution, detergent-stability being an essential prerequisite to purification and crystallisation. The latter approach locks the receptor in a particular conformation which allows successful crystallization as described in WO2008/114020 and WO2009/071914, and also has the advantage that the crystal structure of the GPCR bound to ligands that bind only very weakly can be determined (12,13). As described in WO2009/071914 it was found that stabilising mutations identified in one GPCR could be transferred to another GPCR by aligning the amino acid sequence thus generating a second GPCR with increased stability. However the positions of the stabilising mutations were not located in a common motif or region but instread scattered throughout the GPCR (the turkey β1-adrenergic receptor, human adenosine receptor, rat neurotensin receptor and human muscarinic receptor).
In recent years most success in obtaining crystal structures of membrane proteins has been for bacterial proteins (14) since these are easier to overexpress using known techniques in Escherichia coli than eukaryotic membrane proteins (15, 16) and are more likely to exhibit stability in detergent solution. In contrast eukaryotic membrane proteins often have poor stability in detergent solutions which severely restricts the range of crystallisation conditions that can be explored. Although the structures of over 300 unique polytopic integral membrane proteins have been determined (blanco.biomol.uci.edu/), less than 10% are eukaryotic and approximately half were purified from natural sources and are stable in detergent solutions.
Transmembrane transporters are similar to GPCRs because they exist in at least two distinct conformations, with the substrate binding site accessible to either the extracellular environment (outward-open) or to the cytoplasm (inward-open), with a number of potential intermediate occluded states where the substrate cannot dissociate to either side of the membrane (40). Indeed, the structures of many bacterial transporters have been determined that fit into the above scheme and, at least in the case of Mhp1 (41) and LeuT (26) different conformations of the same transporter have also been described. Transmembrane transporters have been less widely studied than GPCRs but nevertheless are highly relevant in human physiology and disease. They represent valuable targets in drug discovery and development of therapeutics, for example the monoamine transporters are key targets for therapeutic intervention in a wide range of CNS disorders and as primary targets for drugs of abuse such as cocaine and amphetamines (17, 18). Two of the most widely prescribed drugs fluoxetine (Prozac) and omeprazole (Prilosec) target membrane transporters, and there is a need in the art to understand further the structure and function of transmembrane transporters and their role in disease to meet the demand for new therapies targeting CNS disorders.
Current methodologies for the crystallisation of transporters have relied on the identification of those transporters that are sufficiently stable for purification and crystallisation (19) which has allowed the structure determination of many transporters from different families, but the majority of the structures are of bacterial proteins (20). To fully understand inhibitor binding and the mechanism of transport of the mammalian transporters it is essential to determine their structures. However the mammalian transporters are difficult targets for structural studies due to low levels of functional expression and only a proportion of the expressed protein is correctly folded (21, 22). Heterologous expression of the cocaine-sensitive rat serotonin transporter (SERT), GABA (GAT) and norepinephrine (NET) is possible in baculovirus systems however functional expression levels are low and only a proportion of the expressed protein is correctly folded according to the binding of radiolabelled inhibitors (23, 24).
The SLC6 transporter is a sub-class of the neurotransmitter sodium symporter family (NSS) (25) and plays an important role in regulating neurotransmitter concentrations in the peripheral and central nervous system by re-uptake into the presynaptic nerve termini. Mammalian SLC6 is characterised by 12 transmembrane helices with a large extracellular loop between transmembrane helices 3 and 4 (TM3 and TM4) that is invariably N-glycosylated. Structural studies on this family of transporters has focused on bacterial homologues that are extremely stable, such as LeuT (26).
Lactose permease of E. coli (LacY) has been the focus of a number of studies relating to structure and functionality and the crystal structure has been solved using a mutant (C154G) which renders the protein unable to undergo the structural changes required for transport of sugar across the membrane. The mutation (Cys154 to Gly) causes a more compact structure and decreased conformational flexibility with improved thermostability and little tendency to aggregate. It was also observed that the conformational change caused little or no effect on ligand binding (38). These studies demonstrate that it is possible to obtain a conformationally thermostabilised transporter membrane protein by way of mutation which is suitable for crystallisation. (39). Although these studies are of importance for Lactose Permease they do not comment on the wider problem of how to reliably and efficiently solve the crystal structures of other transporter proteins and GPCRs. Therefore there is a need for a common strategy to produce conformationally stable proteins that have use in crystallisation studies and structure determination. Since the provision of conformationally stable mutants of transporters and/or GPCRs and subsequent screening is time consuming, there is a need for methods that are more efficient and reduce the time taken to produce a conformationally stable mutant.
In view of the difficulty in obtaining high quality crystal structures of mammalian proteins due to poor stability and expression and the low numbers of solved crystal structures, there is a need to produce new methods and techniques that overcome the above-mentioned problems, particularly for membrane transporters which as yet have not benefited from the intensity of research seen for GPCRs.
The cocaine-sensitive rat serotonin transporter (SERT) is a member of the SLC6 sub-class of the neurotransmitter sodium symporter family (NSS) and transports the neurotransmitter serotonin from synaptic spaces into presynaptic neurons thereby terminating the action of serotonin. SERT has been well characterised in terms of physiological function and is the target of many antidepressant medications, however its crystal structure remains to be determined. SERT is known to be unstable in detergent solution (27) making it a challenging target for thermostabilisation studies, however the availability of a high affinity radiolabelled ligand [125I] RTIS5 (β-CIT) allows dicrimination between functional and misfolded protein making the protein a suitable candidate for thermostabilisation studies.
The inventors have applied the conformational thermostabilisation approach recently used for GPCRs to the cocaine-sensitive rat serotonin transporter (SERT) to improve conformational stability and tolerance in detergent. They found that when particular regions in or nearby the alpha helices of the transmembrane regions of integral membrane proteins are mutated, this results in a higher proportion of conformationally stable mutants. This discovery has significance for structure determination of other related membrane proteins since it is possible that mutations in specific regions of a membrane protein can be applied across a range of membrane proteins having similar three-dimensional structures, thereby improving the probability of obtaining conformationally stable mutants for use in crystallisation.